Deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA") are long, threadlike macromolecules, DNA comprising a chain of deoxyribonucleotides, and RNA comprising a chain of ribonucleotides. A nucleotide consists of a nucleoside and one or more phosphate groups; a nucleoside consists of a nitrogenous base linked to a pentose sugar. Typically, the phosphate group is attached to the fifth-carbon ("C-5") hydroxyl group ("OH") of the pentose sugar; however, it can also be attached to the third-carbon hydroxyl group ("C-3 OH"). In a molecule of DNA, the pentose sugar is deoxyribose, while in a molecule of RNA, the pentose sugar is ribose. The nitrogenous bases in DNA are adenine ("A"), cytosine ("C"), guanine ("G"), and thymine ("T"). These bases are the same for RNA, except that uracil ("U") replaces thymine. Accordingly, the major nucleosides of DNA, collectively referred to as "deoxynucleosides" are as follows: deoxyadenosine ("dA"); deoxycytidine ("dC"); deoxyguanosine ("dG"); and thymidine ("T"). The corresponding ribonucleosides are designated as "A"; "C"; "G"; and "U". (By convention, and because there is no corresponding thymidine ribonucleoside, deoxythymidine is typically designated as "T"; for consistency purposes, however, thymidine will be designated as "dT" throughout this disclosure).
The sequence of the nitrogenous bases of the DNA or RNA molecule encodes the genetic information contained in the molecule. The sugar and phosphate groups of a DNA or RNA molecule perform a structural role, forming the backbone of the molecule. Specifically, the sugar moiety of each nucleotide is linked to the sugar moiety of the adjacent nucleotide such that the 3'-hydroxyl of the pentose sugar of one nucleotide is linked to the 5'-hydroxyl of the pentose sugar of the adjacent nucleotide. The linkage between the two pentose sugars is typically via a phosphodiester bond. Based upon this linkage protocol, one end ("terminus") of the nucleotide chain has a 5'-terminus (e.g. hydroxyl, triphosphate, etc.), and the other end has a 3'-hydroxyl group. By convention, the base sequence of a nucleotide chain is written in a 5' to 3' direction, i.e., 5'-ATCG-3' or simply ATCG
DNA and RNA are produced internally by living animals; however, DNA and RNA can be chemically synthesized such that synthetic strands of DNA and RNA can be rapidly and efficiently produced. These strands are typically referred to as "synthetic oligonucleotides" or "oligonucleotides." A widely utilized chemical procedure for the synthesis of oligonucleotides is referred to as the "phosphoramidite methodology." See, e.g., U.S. Pat. No. 4,415,732; McBride, L. and Caruthers, M. Tetrahedron Letters, 24:245-248 (1983); and Sinha, N. et al. Nucleic Acid Res. 12:4539-4557 (1984), which are all incorporated herein by reference. Commercially available oligonucleotide synthesizers based upon the phosphoramidite methodology include, e.g., the Biosearch 8750.TM. and ABI 380B.TM., 392.TM. and 394.TM. DNA synthesizers.
The importance of chemically synthesized oligonucleotides is principally due to the wide variety of applications to which oligonucleotides can be directed. For example, oligonucleotides can be utilized in biological studies involving genetic engineering, recombinant DNA techniques, antisense DNA, detection of genomic DNA, probing DNA and RNA from various systems, detection of protein-DNA complexes, detection of site directed mutagenesis, primers for DNA and RNA synthesis, primers for amplification techniques such as the polymerase chain reaction, ligase chain reaction, etc, templates, linkers, and molecular interaction studies.
The primary structures of DNA and RNA molecules can be depicted as follows: ##STR1##
The key step in nucleic acid synthesis is the specific and sequential formation of internucleotide phosphate linkages between a 5'-OH group of one nucleotide and a 3'-OH group of another nucleotide. Accordingly, in the typical synthesis of oligonucleotides, the phosphate group of an "incoming" nucleotide is combined with the 5'-OH group of another nucleotide (i.e. the 5'-OH group is "phosphorylated" or "phosphitylated"). These groups must be capable of actively participating in the synthesis of the oligonucleotides. Thus, the 5'-OH groups are modified (typically with a dimethoxy trityl ("DMT") group) such that an investigator can introduce two such nucleotides into a reaction chamber and adjust the conditions therein so that the two nucleotides are properly combined; by a series of successive such additions, a growing oligonucleotide having a defined sequence can be accurately generated.
The four bases of the nucleosides, adenine, thymine (uracil in the case of RNA), guanine and cytosine, include moieties which are chemically reactive (e.g., exocyclic amino groups). These groups must be "temporarily" protected, i.e. the protecting groups must be capable of blocking any reactive sites on the base until after the oligonucleotide synthesis is completed; after such synthesis is completed, these groups must also be capable of being removed from the bases such that the biological activity of the oligonucleotide is not affected. Without protecting the amino groups of dA, dC, dG (or the corresponding ribonucleotides in the case of RNA synthesis), undesirable and/or less useful material will be synthesized. Thymine (T), which does not have an amino group, does not typically require a protecting group. The traditional amino protecting group for dA is a benzoyl group ("bz"); for dC, a bz group or an isobutyryl group ("ibu"); and for dG, ibu. It is conventional to indicate which protecting group is being utilized as follows: dA.sup.bz, indicating that a benzoyl protecting group is being used to protect the adenine exocyclic amino group.
In order to maintain an additional degree of control over oligonucleotide synthesis, it is necessary to initiate the synthesis of the oligonucleotides from an insolubilized starting point. I.e., the oligonucleotide is synthesized while "tethered" to an appropriate solid support. This protocol increases the speed and convenience of the synthesis as compared to solution-based oligonucleotide synthesis. Accordingly, the synthesis of oligonucleotides requires, as a final step or steps, the removal of protecting groups from the oligonucleotide ("deprotection") and release ("cleavage") of the oligonucleotide from a solid support material--such steps are required in order to generate a biologically useful oligonucleotide.
"Deprotection" is defined as the process and the time necessary for removal of protecting groups incorporated into the nucleotides; "cleavage" is defined as the process and the time necessary for removal of the synthesized oligonucleotide from a solid support material onto which the nucleotides have been attached.
Deprotection of dG is the rate limiting step for oligonucleotide deprotection. By convention, those in the art will focus on the time necessary to deprotect deoxyguanosine incorporated into the oligonucleotide. With respect to side-product formation (i.e. the formation of undesirable materials), deoxycytidine is particularly vulnerable thereto. Accordingly, it is generally considered useful to monitor side-product formation relative to dC in an effort to ensure that side-product formation is avoided or minimized.
Deprotection and cleavage are typically accomplished utilizing ammonia. Because of the widespread use of ammonia as a deprotection and cleavage reagent, it is conventional to compare other such reagents with ammonia in terms of the times necessary for deprotection and cleavage. Deprotection and cleavage by means of ammonia can require several days at room temperature, or between about 6 and 17 hours at 55.degree. C. Typically, cleavage occurs within about 1 hour at room temperature, the remainder of the time being devoted to deprotection. The synthesis of a typical oligonucleotide (i.e. about 20-25 nucleotides in length) can be synthesized in about 2 hours; approximately six minutes is required to add one nucleotide to the insolubilized and growing nucleotide chain using most commercially available synthesizers. Relative to the time required to synthesize an oligonucleotide, the amount of time necessary to deprotect and cleave the oligonucleotide using ammonia can be excessive.
The use of different protection groups allows for a shortening of the time required for deprotection and cleavage. It has been reported that dimethylformamidine ("DMF") is a useful protection group for dG and dA. See Vu, Hugnh et al. "Fast oligonucleotide deprotection phosphoramidite chemistry for DNA synthesis". Tetrahedron Letters, 31:7269-7272 (1990) which is incorporated herein by reference. As described, oligonucleotides including such protection groups require 1 hour at 55.degree. C. in aqueous ammonia, or 8 hours at room temperature in aqueous ammonia, for deprotection and cleavage. It has also been reported that phenoxyacetyl ("PAC") is a useful protection group for dG and dA. See, EPO Published Application 0241363 A1, "Derives de nucleotides et leur utilization pour 1a synthese d'oligonucleotides", Molko, Didier et al (1987) which is incorporated herein by reference. As described, oligonucleotides including this protection group require between 2 to 8 hours at room temperature in aqueous ammonia for deprotection and cleavage.
An alternative approach to reduce the time required for deprotection and cleavage is to use other deprotection and cleavage reagents alone or in conjunction with ammonia. It has been reported that a reagent comprising a lower alkyl alcohol, water and a non-nucleophilic hindered alkylamine containing from 3 to 6 carbon atoms provides for cleavage within 1-2 hours at room temperature, followed by deprotection in about 20 to 60 minutes at about 80.degree. C. to about 90.degree. C. See EPO Published Application 0323152 A2, "Methods of Synthesizing Oligonucleotides", Woo, Sam Lee, et al (1989) (hereafter "Woo") which is incorporated herein by reference. Treatment of oligonucleotides with a 1:1 mixture (v/v) of n-butylamine/methanol for deprotection and cleavage has been described. See Weber, H. and Khorana, H. G. "CIV. Total synthesis of the structural gene for an alanine transfer ribonucleic acid from yeast. Chemical synthesis of an icosa-deoxynucleotide corresponding to the nucleotide sequence 21 to 40." J. Mol. Biol. 72:219-249 (1972) (hereinafter "Weber") which is incorporated herein by reference. However, this reagent is reported to have led to the formation of deoxycytidine side-products. Therefore, and as further described, oligonucleotides not containing cytidine where treated with a 1:1 mixture (v/v) of n-butylamine and methanol at room temperature for 2 days, and for oligonucleotides containing cytidine, treatment first included ammonia at room temperature for 2 days, (to avoid the formation of deoxycytidine side-products) followed by a 1:1 mixture (v/v) of n-butylamine and methanol at room temperature for 2 days.
The following is a brief summary of the described times necessary for deprotection and cleavage for the foregoing protecting groups and reagents:
______________________________________ DEPROTECTION/CLEAVAGE TIMES REAGENT TIME (HOURS) TEMP (.degree.C.) ______________________________________ Ammonia 6-17 55 Ammonia &gt;24 R.T. DMF/Ammonia 2 55 DMF/Ammonia 8 R.T. PAC/Ammonia 8 R.T. Woo* 1-3 R.T.; 80/90 Weber** 2 days R.T. Weber*** 4 days R.T. ______________________________________ *mixture of lower alkyl alcohol, water, nonnucleophilic hindered alkylamine containing from 3-6 carbon atoms; 1-2 hours at R.T. (cleavage) and 20-60 minutes at 80-90.degree. C. (deprotection) **nonC containing oligonucleotides: 1:1 mixture (v/v) nbutylamine/methanol. ***oligonucleotide including C: ammonia (2 days) followed by nbutylamine/methanol (2 days). R.T. = room temperature
While certain of the foregoing reported reaction times for cleavage and deprotection are less than that of ammonia alone, it is evident that relative to the time necessary to synthesize an oligonucleotide, much time is devoted to deprotection and cleavage.
What is needed, given the rapidity in which oligonucleotides can be synthesized, is a reagent that can both rapidly remove the variety of available protection groups, and cleave the oligonucleotide from the support, with minimal to no side-product formation. From a practical perspective, it is preferred that such a reagent be useful across various temperatures, such that, in conjunction with the step of heating, the rapidity at which such a reagent can both deprotect and cleave an oligonucleotide is increased.